All animal experimental procedures in this protocol were approved by the Wuxi 9th People’s Hospital Institutional Review Board (no. KT2019019). A total of 70 skeletally mature male Sprague-Dawley rats, aged 12 weeks, were purchased from Yangzhou University (Yangzhou, China). Rats were housed under a 12-hour light/dark cycle in a temperature (22 ± 1˚C) and humidity (55 ± 5%) controlled room. Standard rodent chow and water were provided ad libitum. The health status of each rat was monitored throughout the experiment by animal veterinary technicians. The rats were free of all viral, bacterial, and parasitic pathogens during the experimental schedule.
Joint Immobilization and Remobilization
The right knee joints of rats were immobilized according to the method described in previous studies [15, 16]. Briefly, after anesthesia by intraperitoneal injection of sodium pentobarbital, a lateral parapatellar arthrotomy of the knee was performed. The patella was moved medially and the knee joint was flexed to expose the femoral condyles. Next, two 1.5-mm x 1.5-mm cortical bone defects were created at the non-cartilaginous portions of the medial and lateral femoral condyles to mimic an intra-articular fracture with hematoma formation. Then, the anterior and posterior cruciate ligaments were transected, and the joint was hyperextended to 45˚ to disrupt the posterior capsule. Finally, a 0.8 mm-diameter Kirschner wire (K-wire) was drilled from the tibia to the femur and curved at both ends to immobilize the knee joint at approximately 160˚ flexion (angle between tibia and femur is approximately 20˚). The skin was then closed with 4.0 silk sutures. Following surgery, rats were allowed to move freely with all four limbs. Five rats were sacriﬁced at each of five time points after immobilization (1 week, 3 weeks, 6 weeks, 9 weeks and 12 weeks; n = 5 each; Fig. 1a). Six weeks after the initial surgery of immobilization, five additional rats underwent a second operation on their right knee to remove the K-wires and were allowed to move freely (remobilization) for 3 weeks (Fig. 1b). For the sham-operation, the right lateral parapatellar arthrotomy of knee was performed, but the tibia and femur were not fixed with K-wire. For all surgical groups, the contralateral (non-operated side) knee joints were used as controls .
An additional thirty rats were randomly divided into six groups (n = 5 each; Fig. 1b, c) to investigate the effect of LY2157299 (Selleck, Houston, USA) on joint contracture. The knees were immobilized or remobilized using the same methods as described above. After immobilization or remobilization, the rats were intraperitoneally injected with LY2157299 daily at a dose of 10 mg/kg body weight or an equivalent volume of vehicle (DMSO and PBS) according to the experimental protocol (Fig. 1c). Rats were all euthanized 9 weeks after surgery.
Measurement of ROM
To assess joint contracture, passive knee extension ROM was measured according to the methods described in the previous study . Briefly, rats were euthanized by carbon dioxide (CO2) inhalation. After the skin of the hindlimbs and K-wire were removed, rats were placed in a neutral spine position and the femur was manually fixed at a hip flexion of 90˚. Then, a knee extension moment of 14.6 N/mm was applied by a pulley and weight to stretch the knee joint to reach its physiological restriction while avoiding damage to the soft tissue peripheral to the joint. The angle was measured by an arthrometer between femur and tibia and considered as passive extension ROM.
Histochemistry and Immunohistochemistry
After ROM measurements, the knee joints of rats were resected and kept at flexion of 90˚ and fixed in 10% buffered formalin at 4˚C for 48 hours. The joints were then decalcified in 10% ethylenediaminetetraacetic acid (EDTA, pH 7.4) for 4 weeks at room temperature. Specimens were dehydrated in graded alcohol and embedded in parafﬁn. A series of 5.0-μm sagittal-oriented sections were cut at the medial condylar region using a microtome. Sections were deparafﬁnized and processed for hematoxylin and eosin (H&E) and Masson trichrome staining.
Immunohistochemistry was performed on the remaining sections. Dewaxed parafﬁn sections were heated to 99˚C for 20 minutes in Target Retrieval Solution (Beyotime, P0086) for antigen retrieval followed rehydration. After washing three times with PBS, the sections on slides were incubated with primary monoclonal antibodies against rat IL-1β (abcam, ab9722), p-Smad2/3 (abcam, ab63399), α-SMA (abcam, ab124964), collagen I (abcam, ab34710), collagen III (abcam, ab7778) overnight at 4 °C in a humidiﬁed chamber. Following a washing step, all slides were incubated with secondary antibodies in blocking solution for 1 hour at room temperature. Finally, the sections were stained with 3,3'-diaminobenzidine for 1 minute and counterstained with hematoxylin. The slides were assessed under an optical microscope (DP75; Olympus Corporation, Tokyo, Japan), and the observer was blinded to which specimen was examined.
Histological Assessmentof Joint Capsules
The synovial length and joint capsule thickness were measured with the H&E-stained sections as described in previous studies [2, 3].
Quantification of Joint Capsule Collagen Density
To elucidate the ﬁbrotic changes in the posterior joint capsules, collagen was quantified using the method published by Kaneguchi et al . To assess the deposition of collagen, Masson trichrome stained sections of the posterior joint capsule were photographed at 200× magniﬁcation with a light microscope (DP70; Olympus Corporation, Tokyo, Japan). The percentage of blue area in the total joint capsule area from each slice represented collagen density and was quantified using ImageJ software (National Institutes of Health, Bethesda, MD).
Enzyme-linked Immunosorbent Assay (ELISA)
Blood samples from rats at each time point after joint immobilization (1 week, 3 weeks, 6 weeks, 9 weeks and 12 weeks) and remobilization (3 weeks) were collected and centrifuged at 3000 rpm for 10 min at 4°C. Control samples were obtained from the sham group. All serum samples were collected and stored at -80˚C until analysis. The concentrations of active TGF-β1 and IL-1β in the serum at all times points were measured using ELISA development kits (Mmbio, China) according to the manufacturer’s instructions.
Gene expression analysis
We extracted total RNA from parafﬁn sections according to the methods of previous studies [2, 19]. In brief, Twenty-micrometer sagittal sections of the lateral side of the knee were cut using a microtome and the posterior joint capsule was isolated with forceps under a stereomicroscope. RNeasy Extract Kit (Solarbio, Beijing, China) was used to extracted total RNA following the manufacturer’s protocol. cDNA was prepared by reverse transcription using the HiScript II First-strand synthesis system (Vazyme biotech, Nanjing, China) according to the manufacturer’s instructions and stored at -20 ˚C until used for quantitative real-time polymerase chain reaction (qRT-PCR). qRT-PCR amplification was performed using the SYBR Premix Ex TaqTM kit (Takara) in a 20 mL reaction containing 0.4 mL of each primer, 0.4 mL SYBR Green Dye and 2 mL of cDNA. The primer sequences are as follows: IL-1β, TGTGATGTTCCCATTAGAC, reverse primer AATACCACTTGTTGGCTTA; TGF-β1, forward primer CCCACTGATACGCCTGAG, reverse primer GACTGATCCCATTGATTTCC; α-SMA, reverse primer CATCCGACCTTGCTAACG, reverse primer TCCAGAGTCCAGCACAATAC; COL1A1, forward primer ATGCCATCAAGGTCTACTGC, reverse primer AATCCATCGGTCATGCTCT; COL3A1, forward primer CCACCCTGAACTCAAGAGC, reverse primer TGAACTGAAAGCCACCATT; β-actin, forward primer CACCCGCGAGTACAACCTTC, forward primer CCCATACCCACCATCACACC. qRT-PCR was carried out on a Roche Light Cycler 480II with the following program: 95°C for 30 sec followed by 40 cycles of 95 °C for 5 sec and 58°C for 34 sec. The relative gene expression level was calculated with 2-DDCt method using β-actin as an internal control.
All data are presented as mean ± standard deviation (SD). Statistical analyses were performed using SPSS software (version 25.0; IBM, Armonk, NY, USA) and all figures were created using GraphPad Prism 8.0 (GraphPad Software, Inc.). We performed comparisons using one-way analysis of variance (ANOVA) and Tukey’s post-hoc test to determine signiﬁcance between groups. For all tests, probability (p) values < 0.05 were considered statistically signiﬁcant and marked as *.